17 Seafood Enzymes 385plex is locked. The resolution of rigor mortis is due
to structural decay elsewhere in the muscular struc-
ture, as will be discussed later.
ENZYMATIC DEGRADATION OF
TRIMETHYLAMINE-N-OXIDE
The substance trimethylamine-N-oxide (TMAO) is
found in all marine seafood species but occurs in
some freshwater fish as well (Anthoni et al. 1990,
Parab and Rao 1984, Niizeki et al. 2002). It contri-
butes to cellular osmotic pressure, as previously de-
scribed, but several other physiological functions of
TMAO have also been suggested. TMAO itself is a
harmless and nontoxic constituent, yet it forms a pre-
cursor of undesirable breakdown products. In seafood
products, TMAO can be degraded enzymatically by
two alternative pathways as described below.
THETRIMETHYLAMINE-N-OXIDEREDUCTASE
REACTION
Many common spoilage bacteria reduce TMAO to
trimethylamine (TMA) by means of the enzyme
trimethylamine-N-oxide reductase (EC 1.6.6.9):
This reaction is catalyzed by trimethylamine-N-
oxide aldolase (TMAOase or TMAO demethylase,
EC 4.1.2.32) but may also to some extent be nonen-
zymatic, catalyzed by iron and various reductants
(Vaisey 1956, Spinelli and Koury 1981, Nitisewojo
and Hultin 1986, Kimura et al. 2002). In most cases
in which significant amounts of formaldehyde and
DMA are accumulated, however, species possessing
TMAOase enzyme activity are involved. The reac-
tion leads to cleavage of a C-N bond and the elimi-
nation of an aldehyde, resulting in the classification
of TMAOase as a lyase (EC 4.1.2.32) and the
IUBMB name aldolase.
DMA is a reactive secondary amine with a milder
odor than TMA. Formaldehyde is highly reactive and
strongly affects the texture of fish meat by making it
tougher, harder, more fibrous, and less juicy, as well
as increasing the drip loss in the thawed products.
The quality changes are associated with a loss in
protein solubility and in particular the solubility of
the myofibrillar proteins. For reviews, see Sikorski
and Kostuch (1982), Hultin (1992), Mackie (1993),
Sikorski and Kolakowska (1994) and Sotelo et al.
(1995).
Formaldehyde can react with a number of chemi-
cal groups, including some protein amino acid resi-
dues and terminal amino groups, resulting in dena-
turation and possibly in the cross-linking of proteins,
both of which are believed to be the cause of the
observed effects on seafood products. The formalde-
hyde concentration in severely damaged seafood
products may reach 240 g/g (Nielsen and Jør-
gensen 2004). These concentrations are generally
considered nontoxic, but may still exceed various
national trade barrier limits.
Whereas TMAO is widespread, TMAOase is only
found in a limited number of animals, many of which
belong to the order of gadiform fish (pollock, cod,
etc.). In gadiform species the highest levels of en-
zyme activity are found in the inner organs (kidney,
spleen, and intestine), while the enzyme activity in
the large white muscle is low. The formation of DMA
and formaldehyde in various tissues of certain non-
gadiform fish, as well as of crustaceans and mollusks,
has also been reported, as reviewed by Sotelo and
Rehbein (2000), although the taxonomic distribution
of the enzyme has not been investigated systemati-
cally. The TMAOase content of gadiform fish ex-
hibits large individual variation, probably due to the
influence of biological factors that have not yet been
adequately studied (Nielsen and Jørgensen 2004).CH 3 CH 3
⏐⏐
OuN⎯CH 3 NADH→N⎯CH 3 NAD H 2 O
⏐⏐
CH 3 CH 3
CH 3 CH (^3) O
⏐⏐I
OuN⎯CH 3 → H⎯N⎯CH 3 H⎯C
⏐ u
CH 3 H
TMA has a strong fishy odor, and TMAO reduc-
tase activity is responsible for the typical off-odor of
spoiled fish. Since TMAO reductase is of microbial
origin, the formation of TMA occurs primarily un-
der conditions such as cold storage that allow micro-
bial growth to take place. TMA is thus an important
spoilage indicator of fresh seafood products. The
formation of TMA is further discussed in the food
microbiology literature (e.g., Barrett and Kwan
1985, Dalgaard 2000).
THETRIMETHYLAMINE-N-OXIDEALDOLASE
REACTION
TMAO is also the precursor of the formation of
dimethylamine (DMA) and formaldehyde: